The herpesvirus group (HSV 1 and 2, VZV, CMV, and EBV) cause serious infections in both normal and immunocompromised hosts. Recently, several inhibitors of viral DNA polymerases have emerged as clinically useful drugs for the treatment of some herpesvirus infections. The DNA polymerases of all the herpesviruses are quite similar but significant differences between them may have important implications for the development of more effective antiviral agents. The HSV DNA polymerase (pol) is an important model for the study of viral DNA replication and the mechanism of antiviral drugs. It has been well-characterized as an enzyme, the gene has been cloned and sequenced, and numerous genetic markers (temperature-sensitive and drug-resistance) have been defined. Recently, the pol gene has been expressed in a heterologous system as a functional product, which permits genetic manipulations of the pol gene to answer important questions about domain structure within pol and its interaction with antiviral agents. The broad, long-term objective of this study is to obtain a detailed understanding of the pol structure, mechanism, and interactions with inhibitors, leading to the rational design of new antiviral agents, possibly active against other homologous viral polymerases. The pol gene is biologically active when expressed in COS-1 cells; it is active enzymatically when expressed in yeast cells and also by in vitro translation in reticulocyte lysate. The effects of mutations introduced into the cloned pol gene can be tested by studying their effects on pol expressed in these heterologous system and by viruses expressing the mutant pol genes. Deletion analysis of the pol gene using the in vitro transcription- translation system will be carried out to define the minimum size of the polypeptide possessing core polymerase activity. This information can be used to express a deleted pol in bacteria to obtain a small enough molecule in large enough quantities to permit crystallization and detailed structural analysis. At least six domains within the polypeptide are conserved among the three prokaryotic and seven eukaryotic structures defining this class. Site-directed oligonucleotide mutagenesis will be carried out in these regions to define the contributions of these regions to enzymatic activity and sensitivity to inhibitors. Specific interaction of the in vitro translated pol with the 65 kD polymerase accessory protein will also be investigated with the pol mutants. The in vitro translated pol and mutants will also be used as targets for affinity-labeling by photoactive nucleotide analogs and specifically designed small DNA fragments.